The present invention relates to primers that specifically hybridize to nucleic acid molecules complementary to the messenger RNA transcribed from genes encoding MAGE tumor-specific antigens or a part thereof or to a complementary strand thereof as well as to diagnostic compositions comprising said antigens. The present invention further relates to methods for detecting disseminated tumor cells employing the primers of the invention as well as methods for preparing a tumor adjuvant vaccine.
Immunotherapeutic approaches have previously been applied to patients with minimal residual disease and could reduce mortality and recurrence associated with distant metastases. Since early occult dissemination of tumor cells is already present in about half of the cancer patients, efficient treatment strategies have to be developed for eliminating residual tumor cells. Many immunocytochemical and PCR-based assays using histogenetic differentiation markers exclusively allow the detection of micrometastatic cells that worsen the prognosis after local tumorectomy without identifying antigens for adjuvant immunotherapy. Limitations of RT-PCR analyses in oncological disorders resulted in the tissue-specific rather than tumor-specific expression of marker genes. Detection of disseminated tumor cells is thus restricted to a few, certain tumor types deriving from similar histological origin. Moreover, tumor cells are only detectable in front of special tissue background, so that most published approaches depend on samples taken from mesenchymal compartments. The presence of ubiquitous transcriptional factors and minimal activation of promotor sequences in background cells may cause illegitimate transcription and reduce specificity and sensitivity due to competing background signals. Furthermore, most assays measure only a single marker gene not considering inter- and intra-individual tumor cell heterogenity.
Several tumor rejection antigens have recently been characterized that are presented on major histocompatibility complex (MHC) class I molecules to autologous cytolytic T-lymphocytes (CTL). The MAGE genes belong to a family of 12 closely related genes with an overall homology of 64-85% and are classified as xe2x80x9ccancer-testis-antigensxe2x80x9d.
The MAGE genes have been employed in the prior art to use primers specific for MAGE 1 to 4, 6 and 12 for the detection of a variety of tumors (WO 95/23874). The specific primers as well as the regions they prime to in the MAGE genes are, however, inappropriate for the diagnosis of disseminated tumor cells in human samples. WO 96/29430 discloses genetic markers for the detection of melanomas and breast cancer cells including occult cancer cells or metastatic cells derived therefrom. In said application, it is further described that a number of markers including MAGE-3 may be employed to detect such tumors. However, methods disclosed in this application are not expected to be suited for the detection of disseminated tumor cells derived from human malignacies of many different histological origins.
Since, in particular, the early reliable detection of disseminated tumor cells in a patient enables the early treatment and/or vaccination in order to prevent recurrence of cancerous growth, the technical problem underlying the present invention was to improve the sensitivity of the prior art methods for detecting such disseminated tumor cells in patients with maligancies of many different histological origins. The solution to said problem is achieved by the embodiments characterized in the claims.
Accordingly, the present invention relates to a primer specifically hybridizing to a nucleic acid molecule complementary to the messenger RNA transcribed from a gene encoding a MAGE tumor-specific antigen or a part thereof or to a complementary strand thereof, said primer being selected from the group of
(i) primers having the same 3xe2x80x2 portion as one of the following groups of primers:
xe2x80x83and;
(ii) primers that overlap with a primer sequence depicted in (a) or (b).
The term xe2x80x9coverlapxe2x80x9d in this context means the overlap of at least one nucleotide.
Accordingly, said primers can hybridize to RNA, preferably mRNA or to DNA, preferably cDNA.
In accordance with the present invention, it was found that the primers having the above-recited 3xe2x80x2 ends are particularly useful in detecting disseminated tumor cells of many different histological origins at a sensitivity level that was unknown from the prior art. Additionally, primers that overlap with the above-recited nucleic acid sequences and that hybridize to MAGE genes have been found useful for the detection of disseminated tumor cells in human samples. Preferably, said primers overlap with the nucleic acid sequences depicted under (a), above. It is further preferred that the primers of the invention have a length of between 15 and 25 nucleotides. With the exception of primers that recognize MAGE-3 and -6, each of said primers specifically detects the expression of only one MAGE gene.
The present invention accordingly provides a means for detecting a cancerous condition at a very early stage of the disease and thus allows the timely treatment of patients where disseminated tumor cells are detected. It was surprisingly found by the present invention that, in contrast to prior art reports, disseminated tumor cells of many different histological origins can be detected using the above-recited primers, even if the analyzed patient has no clinical evidence of disease. It has to be emphasized that the prior art methods and primers as well as the regions of the MAGE genes to which the primers described in the prior art hybridize are not suitable for detecting disseminated tumor cells in samples of patients with malignancies of many different histological origins since the sensitivity of said primers has, in accordance with the present invention, proven not to be high enough for such an analysis. The term xe2x80x9cspecifically hybridizingxe2x80x9d as used in accordance with the present invention is intended to mean that these primers do not cross-amplify nucleic acids from the genes of other MAGE tumor specific antigens in a sensitive RT-PCR. Such RT-PCRs can be devised by the person skilled in the art according to conventional protocols. The specific hybridization occurs under stringent conditions. Such conditions can be devised by the person skilled in the art without undue burden according to conventional protocols; see, e.g., xe2x80x9cNucleic Acid Hybridization, A Practical Approachxe2x80x9d; edited by Hames and Higgins, IRL Press, Oxford 1985.
The primers of the invention are advantageously used in PCR amplification of nucleic acid sequences derived from the desired MAGE genes or fragments thereof. The primers of the invention are devised to avoid the amplification of genomic DNA but to amplify a DNA sequence derived from the reverse transcription of messenger RNA. In this way, the PCR product can be unambiguously correlated with expressed MAGE-genes. PCR may be carried out according to conventional methods. Also, the PCR product may be detected according to protocols that are established in the art.
Preferably, the primer of the invention is a primer depicted in either group (a) or group (b), supra.
The primers of this embodiment have been particularly useful and advantageous in identifying disseminated tumor cells in samples of patients with malignancies of many different histological origins. It is also particularly preferred that primers of group (a) are used as primers for a first round of PCR amplification whereas primers of group (b) are used in a nested primer PCR amplification.
The present invention further relates to a diagnostic composition comprising at least four primers according to the invention wherein said at least four primers hybridize pair-wise to strands of opposite orientation of at least two different nucleic acid molecules wherein strands of one orientation are complementary to the messenger RNA transcribed from the genes of at least two different MAGE tumor-specific antigens.
The diagnostic composition of the invention is particularly useful for carrying out a variety of PCRs and thus for unambiguously identifying the presence of disseminated tumor cells in patients with malignancies of many different histological origins. As has been found in accordance with the present invention, at least two different MAGE-genes from the group of MAGE-1,-2,-3,-4,-6 and -12 should be checked for expression in order to further secure the diagnostic value of the investigation. Preferably, said diagnostic kit comprises at least one pair of primers specific for each of the MAGE-1,-2,-3,-4,-6 and -12 genes. It is further preferred that the analysis of the sample is carried out with the kit of the invention until the expression of at least two different MAGE-genes has been secured.
The bottling of the primers in said kit is effected according to conventional procedures. Preferably, each primer is separately bottled. Alternatively, the primers specific for each MAGE-gene are bottled together with the understanding that primers for a primary and a nested primer reaction are separately bottled.
The present invention further relates to a method of detecting disseminated tumor cells indicative of a cancerous condition in a patient comprising:
(a) carrying out PCR on cDNA obtained from mRNA from one or more patient samples using at least two primers of the invention hybridizing pair-wise to strands of opposite orientation of at least two one cDNA molecule wherein strands of one orientation are complementary to the messenger RNA transcribed from the genes of at least one MAGE tumor-specific antigen; and
(b) detecting one or more PCR products.
The term xe2x80x9ccancerous conditionxe2x80x9d is intended to mean the whole spectrum from the initiation of malignant transformation in single cells to advanced cancer disease including distant solid metastasis.
Preferably, at least four primers specific for at least two MAGE transcripts and complementary strands thereof are employed in the method of the invention.
Additionally, the present invention relates to a method of preparing a tumor adjuvant vaccine comprising the following steps:
(a) carrying out PCR on cDNA obtained from mRNA from one or more patient samples using at least four primers of the invention hybridizing pair-wise to strands of opposite orientation of at least two different nucleic acid molecules wherein strands of one orientation are complementary to the messenger RNA transcribed from the genes of at least two different MAGE tumor-specific antigens;
(b) detecting resulting PCR products; and
(c) using at least one MAGE-gene product, expression of said MAGE-gene being detected as a result of step (b) for preparing an adjuvent tumor vaccine based on MAGE-antigen-derived peptides resembling MHC-restricted T-cell-epitopes, whole MAGE-antigens or fragments thereof, MAGE-transfected host cells, DNA vaccinating strategies using MAGE-encoding nucleotide sequences or any other immunization method based on MAGE-gene products or their coding nucleotide sequences.
This embodiment of the invention relates to the direct application of the positive analysis of the presence of disseminated tumor cells in a human sample. Once said tumor cells have been detected, a suitable antidote for the further proliferation of said tumor cells eventually resulting in tumorous growth should be devised. The method of the present invention provides such an antidote. The tumor adjuvant vaccine can be prepared according to conventional protocols. In particular, it is envisaged to pulse dendritic cells isolated from the patient with peptides derived from the coding sequences of those MAGE-genes detected to be expressed by the disseminated tumor cells of this particular patient using the diagnostic composition of the present invention, these peptides being suited for binding to MHC-molecules of this patient thus resembling MHC-restricted T-cells epitopes. The peptide-pulsed dendritic cells will then be reinjected into the patient and will migrate to the T-cell zones of secondary lymphoid organs where they prime MAGE-specific T-lymphocytes thus resulting in an immune response against MAGE-positive tumor cells.
In a preferred embodiment of the method of the invention, said cDNA is obtained by:
(a) preparing mRNA from one or more samples removed from said patient; and
(b) reverse transcribing said mRNA.
The preparation of said mRNA and the reverse transcription thereof is conveniently effected by conventional procedures.
Reverse transcription of mRNA resulting in the corresponding cDNA may be carried out by random hexanucleotide priming as described in Example 3 by oligo-dT-priming or by specific priming using one or more oligonucleotides complementary to oneor more MAGE-mRNA species. Due to the high degree of sequence homology among different members of the Mage gene family, specific primers for the cDNA-synthesis may be identified that efficiently hybridize to the mRNA of more than one Mage gene and most preferably to all the Mage genes used according to the present invention (Mage-1, -2, -3/6, -4 and -12). Such xe2x80x9cpan-Mage primersxe2x80x9d can be identified and tested according to the state of the art especially represented by computer based sequence analysis and laboratory manuals e.g. Sambrook (Molecular Cloning; A Laboratory Manual, 2nd Edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, N.Y. (1989)) or Hames and Higgins (Nucleic acid hybridization; A practical approach, IRL Press, Oxford/Washington D.C. (1985)).
In an additional preferred embodiment of the method of the invention, said PCR is nested PCR.
As has been found in accordance with the present invention, the nested PCR is a further safeguard for obtaining unambiguous results with regard to the patient""s disease status.
In a further preferred embodiment of the method of the invention, said primers hybridize to 2 to 6 different nucleic acid molecules complementary to the messenger RNA transcribed from the genes of MAGE tumor-specific antigens and to complementary strands thereof.
In a further preferred embodiment of the method of the invention, said MAGE tumor-specific antigens are the MAGE-1, -2, 3, -4, -6 or -12 tumor specific antigens.
Of all MAGE antigens, the above-recited antigens most often appear on tumor cells, as was found in accordance with the present invention, and are therefore particularly indicative of the risk of cancer to develop metastatic tumors.
It is further preferred in accordance with the present invention to remove samples from two different aspiration sites. As was found in accordance with the present invention, the analysis of double-sided aspiration significantly improves sensitivity. It is therefore advisable to use double-sides aspiration in the routine diagnosis of tumor cell dissemination.
In a further preferred embodiment of the method of the invention, said samples to be analyzed are bone marrow aspirates.
As has been surprisingly found in accordance with the present invention, the use of bone marrow aspirates as a source for analyzing the metastatic potential of a patient is more sensitive than using peripheral blood as suggested by the prior art.
Additionally preferred is a method wherein said mRNA preparation comprises the following steps:
(a) immediately lysing the sample in buffer essentially completely avoiding RNA-degradation; and
(b) optionally centrifuging the mRNA obtained in the lysate through a cushion of an RNAse-inhibiting agent, preferably cesium trifluoracetate.
This embodiment of the invention is advantageous since it precludes the loss of mRNA that is, as is well known in the art, sensitive to degradation by exonucleases and therefore precludes loss of sensitivity of the assay method. Further, said method reduces heme concentrations in the RNA-preparation below values inhibiting Taq-polymerase. This is because hemoglobin is removed, high heme concentrations decreasing the sensitivity of the assay method.
The term xe2x80x9cimmediatelyxe2x80x9d means that the sample is lysed within seconds after removal from the patient in an agent depicted under (a). Said agent is preferably a guanidine salt such as guanidine isothiocyanate. After lysis of the sample, said sample is overlaid on a cushion of an RNAse-inhibiting agent depicted under (b). Said agent is preferably a cesium salt such as caesium trifluoroacetate. The mRNA is advantageously obtained by ultracentrifugation through said cushion.
Preferably, the cancerous condition is related to prostate cancer, non-small or small cell lung cancer or sarcoma or malignant melanoma or breast cancer or colorectal cancer. The above enumeration of tumor types is to be understood as a selection of tumors that is recited as preferred examples. Other tumors may be also detected in their early stages in accordance with the present invention.